INTRODUCTION
Commercial bromelain is obtained from the stem and fruit extraction of pineapple (Ananas comosus (L.) Merr.). Bromelain is only found in pineapples. The main composition of bromelain is sulfhydryl proteolytic fraction, peroxidase, acid phosphatase, several protease inhibitors, and organically bound calcium.[ 1 ] Bromelain is widely used in the food, health, pharmaceutical, and cosmetic industries. In the medical and pharmaceutical fields, bromelain is used to modulate tumor growth, and third-degree burns, improve the action of antibiotics, and as oral preparations to treat inflammation, blood coagulation, cancer, and digestion.[ 2 ]
Pineapple is a native tropical plant in Brazil, Bolivia, and Paraguay.[ 1 ] Pineapple fruit in Indonesia is mainly harvested in January, July, and December. Indonesia has 13 provinces of pineapple production centers, including Bogor, Lembang, and Subang districts in West Java Province.[ 3 ] West Java Province produced 228,601 and 250,942 tons of pineapple in 2019 and 2020, respectively,[ 4 ] which increased pineapple production every year. Subang district accounts for 90% of pineapple production in West Java Province.[ 5 ] Its because the altitude of Subang district is 0-1500 m above sea level,[ 6 ] which was suitable for pineapple growth.[ 7 ] Pineapple harvesting always produces waste, including peel, core, and crown. Bromelain is known to spread throughout pineapple. The amount of bromelain in pineapple peel, crown, and leaves is less than that in pineapple stems.[ 1 ] The difference in the pineapple part and the growth place affects the bromelain character.[ 8 ] This study used 5-month old pineapple fruit collected from Subang district, West Java Province, Indonesia. The novelty of this study was crude bromelain isolation from pineapple peel, core, and crown along with its characterization and evaluation of enzyme activity. The pineapple peel, core, and crown were choosen because in Indonesia these parts were agricultural waste and have not been utilized properly. This study was conducted because there has been no study on the character and protease activity of crude bromelain isolated from pineapple peel, core, and crown, which are influenced by the place of growth and the part of the fruit used.
MATERIALS AND METHODS
Materials
The 5-month pineapple fruit was collected from Subang district, West Java Province, Indonesia. The plant was identified by the Plant Taxonomy Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia with No. 33/HB/01/2021. All chemical reagents were analytical grade with no less than 99% purity and purchased from Merck, i.e., ninhydrin, sodium hydroxide, hydrated copper sulfate, potassium sodium tartrate, mercuric nitrate, mercurous nitrate, nitric acid, Folin-Ciocalteu reagent, casein , tyrosine, and trichloroacetic acid (TCA).
Extraction and isolation of crude bromelain
The peel, core, and crown were separated, then each part was washed with distilled water, smashed and filtered. Ethanol was added to each filtrate (1: 4) and stored for 8 h at 4°C, then centrifuged at 15,000 rpm for 15 min. The sediment was dried at 30°C.[ 9 , 10 ]
Protein qualitative test of crude bromelain
Millon’s reagent was added to each crude bromelain solution to form a white precipitate. All mixtures were heated to form a reddish-brown color.[ 11 ] Ninhydrin was added to each crude bromelain solution. All mixtures were heated to form a purple-blue color.[ 12 ] Biuret reagent was added to each crude bromelain solution to form a blue-purple color.[ 13 ]
Determination of total protein content of crude bromelain
The casein standard, the three crude bromelains, and the blank, each was added to the Lowry reagent. All solutions were incubated for 5 min, then measured the absorbance at 595 nm.[ 14 ]
Determination of protease activity of crude bromelain
The various concentration tyrosine solution was prepared, then the absorbance was measured at 275 nm. The tyrosine calibration curve was constructed from concentration and absorbance. Each crude bromelain (1 mg/mL) and casein solution (1 mg/mL) were incubated at 30°C for 30 min. The TCA solution was added to each solution, then heated at 90°C for 5 min. All solutions were cooled to room temperature, then measured the absorbance at 275 nm. One CDU (casein digestive units) defined the amount of enzyme that breaks down into 1 g tyrosine in 1 minute at 37°C.[ 6 , 15 ]
Characterization of crude bromelain
Characterization of optimum temperature and pH was conducted on the three crude bromelains. The optimum temperature was determined at various temperatures (30, 40, 50, 60, and 70°C), while the optimum pHs was determined at various pH (3, 4, 5, 6, 7, 8, 9, 10, and 11).[ 9 , 10 ]
Determination of kinetic parameters (KM and Vmax ) of crude bromelain
The protease activity of each crude bromelain at various casein concentrations (500-20000 ppm) was carried out to determine the kinetic parameters. Lineweaver-Burk graph was used to determine the Vmax and KM of each crude bromelain.[ 9 ]
Statistical analysis
Data were shown as mean and standard deviation (SD). A one-way analysis of variance was conducted to analyze the data statistically. Data included the results of extraction, determination of total protein content, protease activity , characterization, and kinetic parameters of crude bromelain from three different parts of pineapple fruit. The values were considered statistically significant at p -value <0.05.
RESULTS
Extraction of crude bromelain
The results of crude bromelain extraction from the peel, core and crown of pineapple fruit were shown in Table 1 . All crude bromelain was tasteless, light-yellow powder, and slightly pineapple fragrance.
Table 1: Crude bromelain yield
Protein qualitative test of crude bromelain
There was a positive result for the qualitative test for crude bromelain extracted from the peel, core, and crown of pineapple fruit. The result was a reddish-brown precipitate when reacted with Millon’s reagent,[ 11 ] purple solution when reacted with ninhydrin reagent,[ 12 ] and blue solution when reacted with Biuret reagent.[ 13 ]
Determination of total protein content of crude bromelain
The isolation method using ethanol precipitation causes insoluble compounds in ethanol will also precipitate. So, it is necessary to determine the total protein content using casein as a standard. The casein calibration curve was shown in Figure 1 and the total protein content in crude bromelain was shown in Table 2 .
Figure 1: Casein calibration curve
Table 2: Total protein content in crude bromelain
Total protein content was determined from linear regression of the casein calibration curve [Figure 1 ], so crude bromelain contains 40.37 – 45.30% b/b protein [Table 2 ], depends on the fruit part.
Determination of protease activity of crude bromelain
Casein was used as the substrate and tyrosine as the product degradation in the determination of the protease activity of crude bromelain. The tyrosine calibration curse was shown in Figure 2 and the protease activity of crude bromelain was shown in Table 3 .
Figure 2: Tyrosine calibration curve
Table 3: Protease activity of crude bromelain
Protease activity was determined from linear regression of the tyrosine calibration curve, as the casein degradation product [Figure 2 ], so crude bromelain have protease activity of 36.930 – 46.775 unit [Table 3 ], depends on the fruit part.
Characterization of crude bromelain
Enzyme activity was affected by temperature and pH.[ 16 ] The results of the protease activity of crude bromelain in various temperatures was shown in Figure 3 , while in various pH was shown in Figure 4 .
Figure 3: Crude bromelain protease activity at various temperature
Figure 4: Crude bromelain protease activity at various pH
The optimum temperature of crude bromelain isolated from peel and core was 55°C, and 35°C for crown crude bromelain [Figure 3 ]. The optimum pH of the three crude bromelains was pH 7 [Figure 4 ].
Determination of kinetic parameters (KM and Vmax ) of crude bromelain
Kinetic parameters were used to determine the speed of enzyme-catalyzed reactions.[ 16 ] The value of Vmax and KM of crude bromelain was determined by plotting the one per velocity versus one per substrate concentration in the Lineweaver-Burk curve [Figure 5 ]. Then, calculate the linear regression to determine Vmax and KM [Table 4 ].
Figure 5: Lineweaver-Burk curve of crude bromelain
Table 4: Kinetic parameter of crude bromelain
The kinetic parameter of crude bromelain was determined from the Lineweaver-Burk curve, the value of Vmax was obtained when x = 0, while the value of KM was obtained when y = 0 [Figure 5 ]. So, Vmax was in the range of 140.85 to 285.71 units and KM was in the range of 15298.59 to 18370.86 ppm [Table 4 ].
DISCUSSION
Extraction of crude bromelain
The 5-month old fruit was chosen because crude bromelain and its proteolytic activity in raw fruit were higher than the ripe fruit.[ 17 ] Bromelain is soluble in water, i.e., 1 mg/mL,[ 18 ] so distilled water was used as a solvent in crude bromelain extraction. In this study, precipitation with ethanol produced a higher yield than ammonium sulfate, which is in accordance with Jiang et al .[ 16 ] Ethanol, as an organic solvent, is used to precipitate crude bromelain. Organic solvents interfere with the hydrophobic interactions, which stabilize the globular protein core but do not break the covalent bonds.[ 19 ] Denaturation due to organic solvents was reversible, so when ethanol was removed by drying at 30°C, all crude bromelain returned to a globular form with protease activity . Precipitation was aimed to concentrate the crude bromelain, thus facilitating the handling of crude bromelain.[ 20 ] The yield of the peel and crown was higher than the core [Table 1 ], because the core contains more water. There was a significant difference (p-value = 3.41 x 10-4 ) in the yield of the three crude bromelains extracted from three different parts of the pineapple. The smallest yield was from the core, while the yield of the peel was the same as the crown.
Protein qualitative test of crude bromelain
Crude bromelain is a hydrolase enzyme, so it has a protein character. A qualitative protein test was carried out to assess the success of extraction, which determine the positive results of crude bromelain from the three parts of the pineapple fruit. A protein qualitative test was carried out using specific reagents observed from color changes, before and after reacting. Millon’s test detects the phenol group on tyrosine, so this reaction is not specific for protein, because it can detect phenolic compounds.[ 11 ] The ninhydrin test detects free amine groups in amino acids and proteins.[ 12 ] The biuret test detects peptide bonds in proteins.[ 13 ] The qualitative tests showed positive results for crude bromelain, which was analyzed as protein, from the peel, core, and crown of pineapple fruit. This result accordance with our previous studies.[ 9 , 10 ] Until now, there is no specific reagent that only detects bromelain.
Determination of total protein content of crude bromelain
Lowry method has a sensitivity of about 0.1-1.0 mg of protein, with a detection limit of 0.01 mg/mL of protein, and is more sensitive (100 times) than the Biuret method.[ 21 ] Casein was used as a standard protein, because of its high purity and ease to obtain. The correlation coefficient of the casein calibration curve was 0.9944 [Figure 1 ]. This value indicated that the response of the instrument was proportional to the concentration,[ 22 ] so the instrument can be used to determine total protein content.
There was 40.367-45.301% of total protein in crude bromelain isolated from three parts of pineapple fruit [Table 2 ]. Different fruit part contains a different percentage of protein content, with the highest protein content being crown pineapple. Those values indicated the presence of other compounds, that are soluble in distilled water and precipitate within the crude bromelain. There was a significant difference in total protein content in the three crude bromelains (p-value = 1.536 x 10-3 ). This result was similar to our previous study,[ 10 ] but higher than Ketnawa et al .[ 23 ] and Mulyono et al .[ 24 ] The difference in total protein content was caused by the protein precipitation process and the extraction solvent. The total protein content increased when phosphate buffer was used as a solvent, due to the presence of salts that increase protein solubility.[ 23 ] In addition, total protein content was influenced by the age of the pineapple fruit, more riper the pineapple, less protein content.
Determination of protease activity of crude bromelain
Tyrosine as the product of casein degradation was used to determine the protease activity of crude bromelain. The correlation coefficient of the tyrosine calibration curve was 0.9994 [Figure 2 ]. This value indicated that the instrument response was proportional to the concentration,[ 22 ] so the instrument can be used to determine the protease activity of crude bromelain. Determination of enzyme activity is important to determine the actual activity of the extracted enzyme by measuring the product formed.[ 19 ]
Crude bromelain converted casein into amino acid products and tyrosine-containing peptide fragments. The more tyrosine released from casein , the stronger the protease activity .[ 25 ] The Protease activity of the three crude bromelains was in the range of 38.32 to 46.78 units [Table 3 ]. There was a significant difference in the protease activity of the three crude bromelains (p-value = 2.69 x 10-9 ). This result was different from other studies. The protease activity of 100 g of Nang Lae and Phu Lae pineapple peel was 90.653 and 118.920 units, respectively.[ 23 ] While its the activity of the liquid extract of pineapple peel and fruit was 4.71 and 4.52 units, respectively.[ 7 ] The protease activity of liquid extract (500 mg/mL) was 0.014 units for the crown, 0.013 units for the core, 0.01 units for the peel, and 0.006 units for the fruit.[ 26 ] The difference in protease activity was influenced by several conditions, such as temperature, pH, substrate concentration, enzyme concentration, and the effect of inhibitors.[ 27 ]
Characterization of crude bromelain
Temperature affects the kinetic energy of enzyme molecules and the rate of reaction. The increased kinetic energy accelerates the vibrational motion, translation, and rotation of enzymes and substrates, thereby increasing the opportunity for the two to interact with each other. At below or above the optimum temperature, the enzyme undergoes a conformational change, so the substrate cannot enter the active site and decrease the enzyme activity.[ 19 ]
The crude bromelains have protease activity in the range of 25-75°C [Figure 3 ]. At 75°C, crude bromelain still had protease activity , due to its produced products. The optimum temperature for crude bromelain isolated from pineapple peel and core was 55°C, while for pineapple crown was 35°C [Figure 3 ]. There was a significant difference in optimum temperature for the three crude bromelains (p-value = 6.89 x 10-7 ). These results were in the optimum temperature range for stem bromelain, i.e., 35-65°C. The wide temperature range was due to the various molecular weights of bromelain.[ 28 ] The higher the molecular weight, will higher the optimum temperature because the bromelain structure is more stable due to more bonds and interactions in bromelain. Various molecular weights are caused by the different isolation methods. In this study, precipitation was carried out using ethanol followed by centrifugation. Ethanol will reduce the solubility of bromelain in water so that bromelain will precipitate.[ 19 ] Centrifugation is carried out to speed up the precipitation and separation process. This result was different from other studies, due to differences in fruit parts, growth conditions and climate, and the bromelain extraction process. The optimum temperature of bromelain from Nigerian pineapple peel was 40°C,[ 26 ] and 50°C for the Nang Lae pineapple, and 60°C for the Phu Lae pineapple.[ 23 ]
At a certain pH, there is a different charge, and affects the electrostatic bond of the enzyme.[ 26 ] There was an increase in protease activity from pH 3 to 5 in all crude bromelain [Figure 4 ]. The highest activity was obtained at pH 7, then the activity decreased at pH 9 to 11. This result was in accordance with the bromelain pH range, i.e., pH 5 to 7.[ 29 ] At pH 7, crude bromelain has the most suitable conformation with the substrate, so it can form the right enzyme-substrate complex and produce the maximum product.[ 19 ] At below or above the optimum pH, there is decreasing activity, because of decreased the enzyme’s electrostatic bonds. At acidic pH, with a high positive charge, the high electrostatic repulsion causes reduced positive charge uptake in crude bromelain.[ 26 ] This result was in accordance with the result of Ketnawa et al. ,[ 23 ] and Mulyono et al .[ 24 ] and Omotoyinbo et al .[ 26 ] The optimum pH was influenced by the nature of the substrate, the concentration and type of buffer, and the presence of reducing agents.[ 29 ]
Determination of kinetic parameters (KM and Vmax ) of crude bromelain
Determination of enzyme kinetic parameters is needed to explain the catalytic process. The Michaelis-Menten constant (KM ) and the maximum reaction rate (Vmax ) was determined from the Michaelis-Menten equation, which was transformed into the Lineweaver-Burk equation,[ 30 ] in the form of linear regression of various casein concentration [Figure 5 ].
KM indicates the affinity of the enzyme for the substrate, and a small KM indicates a high affinity for the substrate. A high-affinity enzyme binds to substrates at low concentrations, so it can convert substrates to products faster and efficiently. High affinity is affected by more catalytic sites than other enzymes.[ 19 ] Crude bromelain extracted from the crown had the best affinity for casein compared to peel and core [Table 4 ]. There was a significant difference in KM in the three crude bromelains (p-value = 3.73 x 10-4 ). Vmax indicates the rate at which the enzyme converts the substrate to product, aand a low Vmax indicates the substrate is rapidly converted to product.[ 19 ] Crude bromelain extracted from the core had the highest rate of converting casein to tyrosine compared to peel and crown [Table 4 ]. There was a significant difference in Vmax in the three crude bromelains (p-value = 4.72 x 10-6 ). This result was different from other studies, such as stem bromelain having a KM of 5.074 mg/mL and Vmax of 0.666 mg/ml. min,[ 31 ] while fruit bromelain has KM of 0.39 mM and Vmax of 0.38 mol/min.[ 32 ] These results indicate that the source and part of the fruit affect KM and Vmax .
CONCLUSIONS
The three crude bromelains from the peel, core and crown of Indonesian pineapple fruit have protease activity with the specific character and kinetic parameters.
Financial support and sponsorship
This study was supported by Kemenristek Dikti Grants in 2021 [grant number 370/E4/AK.04.ALKES/2021].
Conflicts of interest
There are no conflicts of interest.
REFERENCES
1. Hale LP, Gree PK, Trinh CT, James CL. Proteinase activity and stability of natural bromelain preparation. Int Immunopharmacol 2005; 5: 783–93.
2. Maurer HR. Bromelain:Biochemistry, pharmacology and medical use. Cell Mol Life Sci 2001; 58: 1234–45.
3. pertanianku.com A suitable location for growing pineapple Indonesia. Available from:
www.pertanianku.com . Last accessed on 2021 Dec 14.
4. bps.go.id Fruit crop production. Available from:
https://www.bps.go.id/indicator/55/62/2/produksi-tanaman-buah-buahan.htm . Last accessed on 2021 Dec 15.
5. Subang.go.id Subang district official website. Available from:
https://subang.go.id/ . Last accessed on 2021 Dec 15.
6. Jutamongkon R, Charoenrein S. Effect of temperature on the stability of fruit bromelain. Kasetsart J (Nat Sci) 2010; 44: 943–8.6.
7. Reinhardt DHRC, Bartholomew DP, Souza FVD, de Carvalho ACPP, de Padua TRP, Junghans DT, et al. Advances in pineapple plant propagation. Rev Bras Fruitic Jaboticabal 2018; 40: 1–22.
8. Mondal S, Bhattacharya S, Pandey JN, Biswas M. Evaluation of acute anti-inflammatory effect of Ananas comosus leaf extract in rats. Pharmocologyonline 2011; 3: 1312–5.
9. Saptarini NM, Rahayu D, Kusuma SAF.
Protease activity and characterization of bromelain extract of pineapple (Ananas comusus (L.). Merr) crown from Subang, Indonesia. Rasayan J Chem 2019; 12: 2074–81.
10. Saptarini NM, Rahayu D, Herawati IE. Antioxidant activity of crude bromelain of pineapple (Ananas comosus (L.) Merr) crown from Subang district, Indonesia. J Pharm Bioallied Sci 2019; 11: S551–5.
11. Walsh EO An Introduction to Biochemistry London The English Universities Press Ltd 1961.
12. Kaiser E, Colescott RL, Bossinger CD, Cook PI. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal Biochem 1970; 34: 595–8.
13. Rose F. Über die Verbindungen des Eiweiss mit Metalloxyden (On the compounds of albumin with metal oxides). Poggendorfs Annalen der Physik und Chemie 1833; 104: 132–42.
14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein Measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–75.
15. Mohan R, Sivakumar V, Rangasamy T, Muralidharan C. Optimisation of bromelain enzyme extraction from pineapple (Ananas comosus) and application in process industry. Am J Biochem Biotech 2016; 12: 188–95.
16. Jiang L, He L, Fountoulakis M. Comparison of protein precipitation methods for sample preparation prior to proteomic analysis. J Chromatogr A 2004; 1023: 317–20.
17. Pang WC, Ramli ANM, Hamid AAA. Gene expression analysis of fruit bromelain in ripening of Ananas comosus cultivar MD2. Materials Sci Forum 2020; 981: 209–14.
18. sigmaaldrich.com Product Information:Bromelain From Pineapple Stem. Available from https:
//www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/b5144pis.pdf . Last accessed on 2021 Dec 15.
19. Nelson DL, Cox MM Lehninger:Principles of Biochemistry 5th ed New York (NY) WH Freeman 2008.
20. Martins BC, Rescolino R, Coelho DF, Zanchetta B, Tambourgi EB, Silveira E. Characterization of bromelain from
Ananas comosus agroindustrial residues purified by ethanol factional precipitation. Chem Eng Trans 2014; 37: 781–86.
21. Martina V, Vojtech K. A comparison of biuret, lowry, and bradford methods for measuring the egg's proteins. MENDELNET 2015. Available from:
https://mnet.mendelu.cz/mendelnet2015/articles/62_vrsanska_1167.pdf . Last accessed on 2021 Dec 15.
22. ICH Guidelines for method validation:Q2B validation of analytical procedures:Methodology. Available from:
www.fda.gov . Last accessed on 2021 Dec 15.
23. Ketnawa S, Chaiwut P, Rawdkuen S. Pineapple wastes:A potential source for bromelain extraction. Food Bioprod Process 2012; 90: 385–91.
24. Mulyono N, Rosmeilia E, Moi JGP, Valentine BO, Suhartono MT. Quantity and quality of bromelain in some Indonesian pineapple fruits. Int J Appl Biol Pharm Tech 2013; 4: 235–40.
25. Cupp-Enyard C. Sigma's non-specific
protease activity assay –
casein as a substrate. J Vis Exp 2008; 19: 899.
26. Omotoyinbo O, Sanni D. Characterization of bromelain from parts of three different pineapple varieties in Nigeria. J BioSci 2017; 5: 35–41.
27. Saropah A, Jannah A, Maunatin A. Kinetics of enzymatic reaction coarse extracts of cellulotic bacterial enzymes isolated from rice bran. Alchemy 2012; 2: 34–45.
28. Harrach T, Eckert K, Maurer HR, Machleidt I, Machleidt W, Nuck R. Isolation and characterization of two forms of an acidic bromelain stem proteinase. J Protein Chem 1998; 17: 351–61.
29. Dubey R, Reddo S, Murthy NYS. Optimization of activity of bromelain. Asian J Chem 2012; 24: 1429–33.
30. Bala M, Mel M, Jami MS, Amid A, Salleh HM. Kinetic studies on recombinant stem bromelain. Adv in Enzyme Res 2013; 1: 52–60.
31. Herdyastuti N. Isolation and characteristics of crude extract of bromelain enzyme from pineapple stems. J Biol Res 2006; 12: 75–7.
32. Babagana K, Bala M. Comparative study on characterization of bromelain extracted from the stem and fruit of pineapple (Ananas comosus). J Nat Sci Res 2016; 6: 57–62.
